EP3871680B1 - Double-stranded ribonucleic acid inhibiting expression of complement c5 - Google Patents

Double-stranded ribonucleic acid inhibiting expression of complement c5 Download PDF

Info

Publication number
EP3871680B1
EP3871680B1 EP19876529.9A EP19876529A EP3871680B1 EP 3871680 B1 EP3871680 B1 EP 3871680B1 EP 19876529 A EP19876529 A EP 19876529A EP 3871680 B1 EP3871680 B1 EP 3871680B1
Authority
EP
European Patent Office
Prior art keywords
sirna
seq
double
ribonucleic acid
lipid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP19876529.9A
Other languages
German (de)
English (en)
French (fr)
Other versions
EP3871680A1 (en
EP3871680A4 (en
Inventor
Yuta Suzuki
Sotaro Motoi
Yoshinori Takahashi
Kazuhiro Tahara
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eisai R&D Management Co Ltd
Original Assignee
Eisai R&D Management Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eisai R&D Management Co Ltd filed Critical Eisai R&D Management Co Ltd
Publication of EP3871680A1 publication Critical patent/EP3871680A1/en
Publication of EP3871680A4 publication Critical patent/EP3871680A4/en
Application granted granted Critical
Publication of EP3871680B1 publication Critical patent/EP3871680B1/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5015—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3521—Methyl
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to a double-stranded ribonucleic acid (dsRNA) capable of suppressing expression of complement C5.
  • dsRNA double-stranded ribonucleic acid
  • the present invention relates to a double-stranded ribonucleic acid, a lipid complex encapsulating the double-stranded ribonucleic acid, and a pharmaceutical composition comprising the double-stranded ribonucleic acid or the lipid complex.
  • a protein group called complement includes proteins indicated as C1 to C9, and these proteins are successively activated through three different pathways (classical pathway, lectin pathway, alternative pathway) to elicit immune response.
  • the fifth complement component, C5 is cleaved to C5a and C5b by C5 convertase.
  • C5a is called anaphylatoxin, and induces inflammatory response for various cells via C5aR (CD88) and C5L2 (GPR77).
  • C5b sequentially reacts with C6 to C9 to be converted into a membrane attack complex (MAC) as a final product, which causes bacteriolysis to pathogens or cell lysis.
  • the complement system may elicit strong cytotoxicity to host cells if the complement system fails to be suitably controlled or is excessively activated.
  • the complement C5 is known to be associated with various diseases including paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), myasthenia gravis (MG), neuromyelitis optica (NMO), antibody-mediated rejection in kidney transplantation, Guillain-Barre syndrome, antineutrophil cytoplasmic antibody-associated vasculitis (ANCA-associated vasculitis), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), autoimmune encephalitis, IgG4-related diseases, asthma, antiphospholipid antibody syndrome, ischemia-reperfusion injury, typical hemolytic uremic syndrome (tHUS), multifocal motor neuropathy (MMN), multiple sclerosis (MS), thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, habitual abortion, traumatic brain injury, cold agglutinin disease, dermatomyositis, hemolytic uremic syndrome associated with Shigatoxin-producing Es
  • Non Patent Literature 1 paroxysmal nocturnal hemoglobinuria
  • Non Patent Literature 2 atypical hemolytic uremic syndrome
  • Non Patent Literature 3 myasthenia gravis
  • Neuromyelitis optica Non Patent Literature 4
  • antibody-mediated kidney transplant rejections Non Patent Literature 5
  • the anti-C5 monoclonal antibody eculizumab (Soliris (registered trademark)) exhibits high affinity for complement C5, and suppresses excessive activation of the complement through inhibition of cleavage of C5 into C5a/C5b and accompanying formation of a membrane attack complex.
  • eculizumab exhibits inhibitory effect on hemolysis, and thus is known as a therapeutic agent for paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.
  • eculizumab is known as a therapeutic agent for generalized myasthenia gravis (gMG).
  • gMG generalized myasthenia gravis
  • eculizumab is very expensive, and hence development of alternative means applicable to treatment and prevention of complement C5-mediated diseases is desired.
  • RNA interference examples include methods utilizing RNA interference (hereinafter, also referred to as "RNAi").
  • dsRNA double-stranded ribonucleic acid
  • RISC RNA-induced silencing complex
  • Patent Literature 1 WO 2014/160129
  • An object of the present invention is to provide a novel double-stranded ribonucleic acid for suppressing expression of complement C5.
  • the present invention provides the following embodiments ⁇ 1> to ⁇ 14>.
  • a novel double-stranded ribonucleic acid capable of suppressing expression of complement C5
  • a lipid complex encapsulating the nucleic acid in the lipid complex and a pharmaceutical composition comprising the nucleic acid or the lipid complex can be provided.
  • the double-stranded ribonucleic acid according to the present invention can suppress expression of complement C5 to suppress hemolysis, and hence can be applicable as a therapeutic agent for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS).
  • PNH paroxysmal nocturnal hemoglobinuria
  • aHUS atypical hemolytic uremic syndrome
  • genes encoding complement C5 targeted by the double-stranded ribonucleic acid include, but are not limited to, C5 derived from humans, mice, and monkeys.
  • Information on C5 gene sequences is available from public databases including registered sequence information such as GenBank provided by The National Center for Biotechnology Information (NCBI), or can be obtained by designing a primer based on information of a nucleotide sequence for C5 from a closely related animal species followed by cloning therewith from an RNA extracted from a desired animal species.
  • Examples of the sequence of an mRNA transcript corresponding to the target gene human C5 include the sequence of a human C5 mRNA transcript registered as GenBank Accession No. NM_001735.2 (GI: 38016946).
  • the term "C5 gene” herein is not limited to a gene having a particular sequence. For example, naturally-occurring C5 genes with single nucleotide polymorphism can be also included in the term.
  • combination of sense strand and antisense strand is selected from the group consisting of: SEQ ID NO: 159 and SEQ ID NO: 160, SEQ ID NO: 141 and SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID NO: 144, SEQ ID NO: 145 and SEQ ID NO: 146, SEQ ID NO: 147 and SEQ ID NO: 148, and SEQ ID NO: 153 and SEQ ID NO: 154.
  • the combinations respectively correspond to the sequences of siRNA-008-01, siRNA-008-32, siRNA-008-33, siRNA-008-34, siRNA-008-35 and siRNA-008-38 in the present specification.
  • a sense strand and an antisense strand as any one of the combinations (1) to (6) are pairing.
  • Each of the combinations (1) to (6) of a sense strand and an antisense strand includes a region complementary to each other.
  • a double-stranded ribonucleic acid including the combination of the sense strand of SEQ ID NO: 13 and the antisense strand of SEQ ID NO: 14 includes the following complementary strands (dT ⁇ dT at the 3'-terminal is not shown, see Table 1 for more details).
  • combination of sense strand and antisense strand is selected from the group consisting of: SEQ ID NO: 159 and SEQ ID NO: 160, SEQ ID NO: 141 and SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID NO: 144, SEQ ID NO: 145 and SEQ ID NO: 146, SEQ ID NO: 147 and SEQ ID NO: 148, and SEQ ID NO: 153 and SEQ ID NO: 154.
  • the combinations respectively correspond to the sequences of siRNA-008-01, siRNA-008-32, siRNA-008-33, siRNA-008-34, siRNA-008-35, and siRNA-008-38 in the present specification.
  • combination of sense strand and antisense strand is selected from the group consisting of: SEQ ID NO: 159 and SEQ ID NO: 160, SEQ ID NO: 141 and SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID NO: 144, SEQ ID NO: 145 and SEQ ID NO: 146, SEQ ID NO: 147 and SEQ ID NO: 148, and SEQ ID NO: 153 and SEQ ID NO: 154.
  • the combinations respectively correspond to the sequences of siRNA-008-01, siRNA-008-32, siRNA-008-33, siRNA-008-34, siRNA-008-35, and siRNA-008-38 in the present specification.
  • combination of sense strand and antisense strand is selected from the group consisting of: SEQ ID NO: 141 and SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID NO: 144, SEQ ID NO: 145 and SEQ ID NO: 146, SEQ ID NO: 147 and SEQ ID NO: 148, and SEQ ID NO: 153 and SEQ ID NO: 154.
  • the combinations respectively correspond to the sequences of siRNA-008-32, siRNA-008-33, siRNA-008-34, siRNA-008-35, and siRNA-008-38 in the present specification.
  • a double-stranded ribonucleic acid according to an embodiment comprising a sense strand and an antisense strand includes a sense strand of SEQ ID NO: 159 and an antisense strand of SEQ ID NO: 160.
  • the combination corresponds to the sequence of siRNA-008-01 in the present specification.
  • a double-stranded ribonucleic acid according to an embodiment comprising a sense strand and an antisense strand includes a sense strand of SEQ ID NO: 141 and an antisense strand of SEQ ID NO: 142.
  • the combination corresponds to the sequence of siRNA-008-32 in the present specification.
  • a double-stranded ribonucleic acid according to an embodiment comprising a sense strand and an antisense strand includes a sense strand of SEQ ID NO: 143 and an antisense strand of SEQ ID NO: 144.
  • the combination corresponds to the sequence of siRNA-008-33 in the present specification.
  • a double-stranded ribonucleic acid according to an embodiment comprising a sense strand and an antisense strand includes a sense strand of SEQ ID NO: 145 and an antisense strand of SEQ ID NO: 146.
  • the combination corresponds to the sequence of siRNA-008-34 in the present specification.
  • a double-stranded ribonucleic acid according to an embodiment comprising a sense strand and an antisense strand includes a sense strand of SEQ ID NO: 147 and an antisense strand of SEQ ID NO: 148.
  • the combination corresponds to the sequence of siRNA-008-35 in the present specification.
  • a double-stranded ribonucleic acid according to an embodiment comprising a sense strand and an antisense strand includes a sense strand of SEQ ID NO: 153 and an antisense strand of SEQ ID NO: 154.
  • the combination corresponds to the sequence of siRNA-008-38 in the present specification.
  • the antisense strand is substantially complementary to at least a part of an mRNA transcript of a C5 gene.
  • the phrase "substantially complementary” includes not only cases that the antisense strand is completely complement to a part of an mRNA transcript of a C5 gene but also cases that there are one to several acceptable mismatches between the antisense strand and a part of an mRNA transcript of C5 gene.
  • the sense strand is substantially complementary to at least a part of the nucleotide sequence of the antisense strand.
  • substantially complementary includes not only cases that the sense strand is completely complement to a part of the nucleotide sequence of the antisense strand but also cases that there are one to several acceptable mismatches between the sense strand and a part of the nucleotide sequence of the antisense strand.
  • completely complementary may apply to cases when the oligonucleotide of the longer of the sense strand and the antisense strand includes a nucleotide sequence completely complementary to the oligonucleotide of the shorter.
  • the double-stranded ribonucleic acid according to an embodiment may also include a modified nucleotide, as described later (see also Table 1).
  • a modified nucleotide as described later (see also Table 1).
  • nucleotide used herein is intended not only to refer to guanosine-3'-phosphate, cytidine-3'-phosphate, adenosine-3'-phosphate, and uridine-3'-phosphate, but also to encompass various modified nucleotides.
  • double-stranded ribonucleic acid or “dsRNA” herein refers to a ribonucleic acid (RNA) molecule having double-stranded structure including two antiparallel, substantially complementary oligonucleotides, or a complex thereof.
  • double-stranded ribonucleic acids include, but are not limited to, siRNAs (small interfering RNAs).
  • the double-stranded ribonucleic acid according to an embodiment comprises a sense strand and an antisense strand.
  • an mRNA for a C5 gene is cleaved as the target mRNA molecule in an RISC complex, and as a result expression of C5 is suppressed.
  • expression of C5 in cells in a subject is suppressed.
  • the double-stranded ribonucleic acid according to an embodiment can be synthesized, for example, by using a method with chemical synthesis known in the art (e.g., described in Nucleic Acid Research, 35(10), 3287-96 (2007 )) and enzymatic transcription.
  • the double-stranded ribonucleic acid according to an embodiment may include various modifications. Modification can be performed by using a method known in the art. Examples of the modification include sugar modification.
  • sugar modification examples include modification for the ribose moiety constituting ribonucleoside, specifically, substitution or addition at the hydroxy group at the 2'-position, more specifically, 2'-O-methyl-modified nucleotide in which the hydroxy group has been substituted with a methoxy group.
  • Nucleotides represented as lowercase a, u, g, and c in Table 1 are 2'-O-methyl-modified nucleotides, and the sense strand and antisense strand of the double-stranded ribonucleic acid according to an embodiment may each include 2'-O-methyl-modified nucleotide.
  • the double-stranded ribonucleic acid can be modified by inserting an additional nucleotide or nucleotide derivative, which is called overhang, into the 3'-side or 5'-side of a region where the sense strand and the antisense strand are forming a double strand.
  • the double-stranded ribonucleic acid as disclosed herein may include the sense strand and/or the antisense strand including deoxy-thymidine (dT) at the 3'-terminal as SEQ ID NO: 13 and SEQ ID NO: 14, and the sense strand and/or the antisense strand including inverted deoxy-thymidine (idT) as SEQ ID NO: 129.
  • the double-stranded ribonucleic acid as disclosed herein may also include the sense strand and/or the antisense strand including U, A, and so forth, added as an overhang sequence, for example, that including UUUU added at the 3'-terminal of the antisense strand as SEQ ID NOs: 140 and 142.
  • the double-stranded ribonucleic acid can be backbone-modified through modification or substitution of the phosphodiester bond.
  • the modification or substitution of the phosphodiester bond include a phosphorothioate bond.
  • the double-stranded ribonucleic acid as disclosed herein may also include that including neighboring nucleotides connected with a phosphorothioate bond as SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 121.
  • the double-stranded ribonucleic acid according to an embodiment can be introduced into cells in a subject by using a chemical method (e.g., transfection), physical method (e.g., electroporation, microinjection), or biological method (e.g., virus vectors) known in the art.
  • a chemical method e.g., transfection
  • physical method e.g., electroporation, microinjection
  • biological method e.g., virus vectors
  • Transfection is a method of bonding a nucleic acid to a positively charged substance (such as a liposome and polymer) to form a complex followed by allowing cells to incorporate the complex by endocytosis through attracting the complex to negatively charged cell surfaces.
  • Transfection can be performed by using a known method, and can be performed by using a commercially available transfection reagent (e.g., TransIT (registered trademark) series from Takara Bio Inc., Lipofectamine (registered trademark) series from Invitrogen) in a simple manner.
  • Nucleic acid molecules are easily degraded by ribonuclease present in the living body and each nucleic acid molecule itself is a negatively charged polymer, and hence it is difficult for nucleic acid molecules to pass cell membranes, which are also negatively charged.
  • a technique with a Drug Delivery System (DDS) using a lipid complex, polymer, or the like has been developed.
  • a lipid complex encapsulating (I) the double-stranded ribonucleic acid of the invention may comprise (II) a cationic lipid, and (III) at least one lipid selected from the group consisting of neutral lipid, polyethylene glycol-modified lipid (PEG lipid), and sterol.
  • PEG lipid polyethylene glycol-modified lipid
  • sterol lipid-modified lipid
  • examples of the lipid complex herein include, but are not limited to, LNPs (lipid nanoparticles).
  • Examples of the form of a complex formed of a lipid containing a cationic lipid and the double-stranded ribonucleic acid include a complex of the double-stranded ribonucleic acid and a membrane consisting of a lipid monolayer (single molecule) (reverse micelle); a complex of the double-stranded ribonucleic acid and a liposome; and a complex of the double-stranded ribonucleic acid and a micelle.
  • the double-stranded ribonucleic acid may be encapsulated in a fine particle comprising a lipid containing a cationic lipid.
  • the lipid complex according to an embodiment may contain the double-stranded ribonucleic acid in a content of, for example, 0.01 to 50% by weight, 0.1 to 30% by weight, or 1 to 10% by weight to the total weight of the lipid complex.
  • Cationic lipid is an amphiphilic molecule having a lipophilic region including one or more hydrocarbon groups and a hydrophilic region including a polar group to be protonated at specific pH.
  • Examples of the cationic lipid according to an embodiment include, but are not particularly limited to, cationic lipids described in International Publication Nos. WO 2015/105131 , WO 2016/104580 , and WO 2017/222016 , and alternatively a cationic lipid with improved biodegradability described in International Publication No. WO 2016/104580 or WO 2017/222016 can be used.
  • Examples of the cationic lipid according to an embodiment include 1-oxo-1-(undecan-5-yloxy)nonadecan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxononadecan-10-yl-1-methylpiperidine-4-carboxylate, 1-oxo-1-(undecan-5-yloxy)heptadecan-8-yl-1-methylpiperidine 4-carboxylate, 21-oxo-21-(undecan-5-yloxy)heneicosan-10-yl-1-methylpiperidine4-carboxylate, 21-(octan-3-yloxy)-21-oxoheneicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, (Z)-1-((2-buty
  • the lipid complex according to an embodiment contains the above-described cationic lipid in a content of, for example, 10 to 100 mol%, 20 to 90 mol%, or 40 to 70 mol% based on the total lipids contained in the lipid complex.
  • One cationic lipid can be used singly, and mixture of two or more cationic lipids can also be used.
  • the lipid complex according to an embodiment may comprise (I) the above-described cationic lipid and (II) at least one lipid selected from the group consisting of neutral lipid, polyethylene glycol-modified lipid, and sterol, as a lipid component.
  • the lipid complex according to an embodiment may contain the lipid component in a content of, for example, 50 to 99.99% by weight, 70 to 99.9% by weight, or 90 to 99% by weight to the total weight of the lipid complex.
  • neutral lipid refers to a lipid present either as a non-charged form or as a neutral zwitterion at physiological pH.
  • Examples of the neutral lipid according to an embodiment may include dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), dilignoceroylphosphatidylcholine (DLPC), dioleoylphosphatidylcholine (DOPC), sphingomyelin, ceramide, dioleoylphosphatidylglycerol (DOPG), dipalmit
  • the lipid complex according to an embodiment may contain the neutral lipid in a content of, for example, 0 to 50 mol%, 0 to 40 mol%, 0 to 30 mol%, or 0 to 20 mol% based on the total lipids contained in the lipid complex.
  • Examples of the polyethylene glycol-modified lipid (PEG lipid) may include PEG2000-DMG (PEG2000-dimyristyl glycerol), MPEG2000-DMG (MPEG2000-dimyristyl glycerol), PEG2000-DPG (PEG2000-dipalmitoylglycerol), PEG2000-DSG (PEG2000-distearoylglycerol), PEG5000-DMG (PEG5000-dimyristyl glycerol), PEG5000-DPG (PEG5000-dipalmitoylglycerol), PEG5000-DSG (PEG5000-distearoylglycerol), PEG-cDMA (N-[(methoxypoly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxylpropyl-3-amine), PEG-C-DOMG (R-3-[( ⁇ -methoxy-poly(ethylene glycol)-
  • PEG-dialkyloxypropyl examples include PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, and PEG-distearyloxypropyl.
  • the polyethylene glycol-modified lipid may MPEG2000-DMG (MPEG2000-dimyristyl glycerol).
  • MPEG2000-dimyristyl glycerol MPEG2000-dimyristyl glycerol.
  • One polyethylene glycol-modified lipid can be used singly, and mixture of two or more polyethylene glycol-modified lipids can also be used.
  • the lipid complex according to an embodiment may contain the polyethylene glycol-modified lipid in a content of, for example, 0 to 30 mol%, 0 to 20 mol%, 0 to 10 mol%, or 0.5 to 2 mol% based on the total lipids contained in the lipid complex.
  • Sterol is an alcohol having a steroid backbone.
  • examples of the sterol according to an embodiment may include cholesterol, dihydrocholesterol, lanosterol, ⁇ -sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, fucosterol, and 3 ⁇ -[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol).
  • the sterol is cholesterol.
  • One sterol can be used singly, and mixture of two or more sterols can also be used.
  • the lipid complex according to an embodiment may contain the sterol in a content of, for example, 0 to 90 mol%, 10 to 80 mol%, or 20 to 40 mol% based on the total lipids contained in the lipid complex.
  • Combination of lipid components in the lipid complex according to an embodiment is not particularly limited, and examples thereof include combination of the above-described cationic lipid, neutral lipid, and sterol, and combination of the above-described cationic lipid, neutral lipid, polyethylene glycol-modified lipid, and sterol.
  • the lipid complex according to an embodiment encapsulating the double-stranded ribonucleic acid may comprise lipid components of cationic lipid/neutral lipid/polyethylene glycol-modified lipid/sterol, and the mole ratio of the lipids may be, for example, 10 to 99/0 to 50/0 to 10/0 to 50, or 40 to 70/0 to 20/0.5 to 2/20 to 40.
  • the "average particle size" of the lipid complex particle encapsulating the double-stranded ribonucleic acid according to the present invention refers to the Z-average particle size.
  • the average particle size (Z-average) of a lipid complex according to an embodiment encapsulating the double-stranded ribonucleic acid may be, for example, 10 to 1000 nm, 30 to 500 nm, or 30 to 200 nm as measured by using a particle size analyzer (Malvern Panalytical Ltd., Zetasizer Nano ZS), though the average particle size is not particularly limited thereto.
  • the siRNA encapsulation efficiency for a lipid complex according to an embodiment encapsulating the double-stranded ribonucleic acid can be calculated, for example, from the siRNA concentration of a formulation diluted with RNase Free Water, which is assumed as the concentration of siRNA present in the LNP external solution, and the siRNA concentration of the formulation diluted with 1% Triton X-100, which is assumed as the total siRNA concentration of the formulation, where each siRNA concentration is measured by using Quant-iT RiboGreen RNA Reagent (Invitrogen, Cat#R11491) (see also Kewal K. Jain, Drug Delivery System, Methods in Molecular Biology, Vol. 1141: 109-120 ).
  • the encapsulation efficiency calculated in this manner be, for example, higher than 80%, higher than 85%, or higher than 90%. It is preferable that the siRNA encapsulation efficiency for a lipid complex according to an embodiment encapsulating the double-stranded ribonucleic acid be higher than 90%.
  • Examples of methods for encapsulating an effective molecule in a lipid complex include a reverse phase evaporation method, a zwitterion (NaCl) hydration method, a cationic core hydration method, and a method with ethanol and calcium (see, Biomembr., 1468, 239-252 (2000 )).
  • a lipid complex according to an embodiment encapsulating the double-stranded ribonucleic acid can be prepared by using any of these methods known in the art.
  • a lipid complex according to an embodiment encapsulating the double-stranded ribonucleic acid can be prepared by, for example, mixing a lipid solution containing the cationic lipid and at least one lipid selected from the group consisting of neutral lipid, polyethylene glycol-modified lipid, and sterol, and an acidic buffer containing the double-stranded ribonucleic acid.
  • a lipid complex the inside of which is filled with a core of the double-stranded ribonucleic acid and the lipids can be obtained.
  • a lipid complex according to an embodiment encapsulating the double-stranded ribonucleic acid may contain the cationic lipid and at least one lipid selected from the group consisting of neutral lipid, polyethylene glycol-modified lipid, and sterol.
  • a lipid complex according to an embodiment encapsulating the double-stranded ribonucleic acid can be produced by using a method including: a step (a) of mixing a polar organic solvent-containing aqueous solution containing (I) the cationic lipid and (II) at least one lipid selected from the group consisting of neutral lipid, polyethylene glycol-modified lipid, and sterol, and an aqueous solution containing (III) the double-stranded ribonucleic acid to obtain a mixed solution; and a step (b) of reducing the content of the polar organic solvent in the mixed solution.
  • a lipid complex encapsulating the double-stranded ribonucleic acid in a fine particle comprising the lipids can be formed.
  • a lipid complex can be formed by reducing the content of the polar organic solvent in the mixed solution to change the solubility of the lipid component containing (I) the cationic lipid and (II) at least one lipid selected from the group consisting of neutral lipid, polyethylene glycol-modified lipid, and sterol in the polar organic solvent-containing aqueous solution.
  • the polar organic solvent include alcohol such as ethanol.
  • a polar organic solvent-containing aqueous solution containing (I) the cationic lipid and (II) at least one lipid selected from the group consisting of neutral lipid, polyethylene glycol-modified lipid, and sterol dissolved therein is mixed with an aqueous solution containing (III) the double-stranded ribonucleic acid to obtain a mixed solution.
  • concentration of the polar organic solvent in the polar organic solvent-containing aqueous solution is not particularly limited as long as conditions for dissolving lipid molecules are satisfied even after mixing with the aqueous solution containing the double-stranded ribonucleic acid.
  • the concentration of the polar organic solvent in the polar organic solvent-containing aqueous solution in the step (a) can be 0 to 60% by weight.
  • the aqueous solution containing (III) the double-stranded ribonucleic acid is obtained by, for example, dissolving the double-stranded ribonucleic acid in an acidic buffer.
  • the content of the polar organic solvent is reduced by adding water or the like to the mixed solution.
  • a lipid complex can be formed. It is preferred for efficient formation of the lipid complex to rapidly lower the content of the polar organic solvent.
  • the concentration of the polar organic solvent in the final polar organic solvent-containing aqueous solution in the step (b) can be 0 to 5% by weight.
  • the mixed solution obtained in the step (a) may be subjected to dialysis to remove the polar organic solvent and substitute the solvent with a pharmaceutically acceptable medium.
  • the content of the polar organic solvent in the solution decreases during the dialysis, by which a lipid complex can be formed.
  • lipid complex encapsulating the double-stranded ribonucleic acid in the inside of a fine particle can be obtained with high encapsulation efficiency.
  • Examples of the acidic buffer to dissolve the double-stranded ribonucleic acid therein include sulfate buffer, phosphate buffer, phthalate buffer, tartrate buffer, citrate buffer, formate buffer, oxalate buffer, and acetate buffer.
  • Examples of the solvent to dissolve the lipids therein include polar organic solvent such as alcohol, and the solvent may be, for example, ethanol, isopropanol, chloroform, or tert-butanol.
  • a pharmaceutical composition containing a double-stranded ribonucleic acid according to an embodiment or a lipid complex encapsulating the double-stranded ribonucleic acid can be provided.
  • the pharmaceutical composition can contain a pharmaceutically acceptable carrier in addition to a double-stranded ribonucleic acid according to an embodiment or a lipid complex encapsulating the double-stranded ribonucleic acid.
  • Examples of the pharmaceutically acceptable carrier include liquid or solid fillers, diluent, excipients, production aids, and solvent-encapsulating materials.
  • the pharmaceutical composition according to an embodiment may be, for example, in the form of powder obtained by removing solvent through freeze-drying or the like, or in the form of liquid.
  • a pharmaceutical composition according to an embodiment may be a powder composition containing a lipid complex according to any of the above-described embodiments.
  • the powder composition may be prepared by removing solvent from a composition in the form of liquid (dispersion), for example, through filtration or centrifugation, or prepared by freeze-drying the dispersion.
  • the pharmaceutical composition is in the form of powder, the pharmaceutical composition can be suspended or dissolved in a pharmaceutically acceptable medium before use and used as an injection.
  • a pharmaceutical composition according to an embodiment may be a liquid composition containing a lipid complex according to any of the above-described embodiments and a pharmaceutically acceptable medium.
  • the pharmaceutical composition in the form of liquid, the pharmaceutical composition can be directly used as an injection, or suspended or dissolved in a pharmaceutically acceptable medium and used as an injection.
  • RNAi By administering a pharmaceutical composition according to an embodiment to a subject in need thereof, expression of complement C5 in the subject can be inhibited through RNAi.
  • the "subject in need thereof” refers to a subject presenting with a disease or disorder associated with expression or activity of the C5 gene, or a subject determined to have a high risk of development thereof.
  • the double-stranded ribonucleic acid can inhibit expression of complement C5, and hence a complement C5 inhibitor or pharmaceutical composition containing the double-stranded ribonucleic acid or a lipid complex encapsulating the double-stranded ribonucleic acid can be useful for treating paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS).
  • PNH paroxysmal nocturnal hemoglobinuria
  • aHUS atypical hemolytic uremic syndrome
  • a method for treating paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome including a step of administering a therapeutically effective amount of a pharmaceutical composition according to an embodiment, is disclosed.
  • use of the double-stranded ribonucleic acid according to an embodiment or a lipid complex encapsulating the double-stranded ribonucleic acid according to an embodiment for producing a therapeutic drug for paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome is disclosed.
  • the double-stranded ribonucleic acid according to an embodiment or a lipid complex encapsulating the double-stranded ribonucleic acid according to an embodiment for use in a method for treating paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome is provided.
  • the double-stranded ribonucleic acid according to an embodiment or a lipid complex encapsulating the double-stranded ribonucleic acid according to an embodiment in the lipid complex can be used singly or in combination with another agent or composition in a therapeutic method.
  • the double-stranded ribonucleic acid according to an embodiment or a lipid complex encapsulating the double-stranded ribonucleic acid according to an embodiment may be administered simultaneously with or separately from administration of another agent.
  • Such combination therapy includes combined administration (two or more agents are contained in one formulation or different formulations) and separate administration (e.g., simultaneous or sequential).
  • the double-stranded ribonucleic acid according to an embodiment or a lipid complex encapsulating the double-stranded ribonucleic acid according to an embodiment may be administered prior to or sequentially after the accompanying therapeutic method.
  • the subject to administer a pharmaceutical composition containing the double-stranded ribonucleic acid according to an embodiment or a lipid complex encapsulating the double-stranded ribonucleic acid according to an embodiment is not limited, and, for example, the subject can be humans or non-human mammals (such as monkeys, mice, rats, rabbits, cows, horses, goats).
  • the method for administering a complement C5 inhibitor or pharmaceutical composition containing the double-stranded ribonucleic acid according to an embodiment or a lipid complex encapsulating the double-stranded ribonucleic acid according to an embodiment to a subject is not limited, and can be appropriately determined by one of ordinary skill in the art (e.g., physicians) in accordance with the health condition of a subject, the degree of a disease, the type of an agent to be used in combination.
  • the mode of administration of a pharmaceutical composition according to an embodiment is not particularly limited, and may be parenteral administration, and examples thereof include intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, and intrathecal administration.
  • a pharmaceutical composition according to an embodiment can be administered in an amount enough to inhibit complement C5 depending on the mode of administration.
  • the dose of a pharmaceutical composition according to an embodiment may be, for example, 0.01 mg to 100 mg, or 0.1 mg to 50 mg, or 0.3 mg to 10 mg, per kg body weight of a subject.
  • first and second are used to represent various elements, it is to be understood that such elements should not be limited by the terms themselves. The terms are used only to distinguish one element from another element, and, for example, it is acceptable without departing from the scope of the present invention to express a first element as “second element” and to express a second element as “first element”, similarly.
  • Sense strands and antisense strands listed in Table 2 were synthesized by using the phosphoramidite method, and then annealed to synthesize double-stranded nucleic acids (GeneDesign, Inc.). Abbreviations in the sequences are as shown in Table 1. Each double-stranded nucleic acid synthesized had a hydroxy group instead of a phosphate group at each 3'-terminal.
  • RNAiMax each of the double-stranded nucleic acids listed in Table 2 in combination with the transfection reagent Lipofectamine RNAiMax (from Invitrogen, catalog number: 13778150) was diluted with an Opti-MEM medium (from Gibco, catalog number: 31985062) to prepare siRNA/RNAiMax mixed solution with a final concentration of 3 nM double-stranded nucleic acid and 0.3% RNAiMax.
  • the siRNA/RNAiMax mixed solution was aliquoted into 20 ⁇ L portions in wells of a 96-well culture plate, and Hep3B cells (obtained from ATCC) as cell lines derived from human liver cancer were seeded in each well at 20000 cells/80 ⁇ L/well, and cultured under conditions of 37°C and 5%CO 2 overnight.
  • a template lysate for real-time PCR was prepared by using a CellAmp (registered trademark) Direct RNA Prep Kit for RT-PCR (Real Time) (from Takara Bio Inc., catalog number: 3732) and Proteinase K (from Takara Bio Inc., catalog number: 9034) in accordance with a protocol provided by Takara Bio Inc.
  • cDNA was prepared by using a PrimeScript (registered trademark) RT Master Mix (Perfect Real Time) (from Takara Bio Inc., catalog number: RR036A) in accordance with a protocol provided by Takara Bio Inc. Further, Ct values were measured for the target gene human C5 and the endogenous control gene human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) by using an EagleTaq Universal Master Mix (ROX) (from Roche Diagnostics K.K., catalog number: 07260296190) and a TaqMan probe (from Applied Biosystems, C5: Hs00156197_m1; GAPDH: Hs02758991_g1) with an ABI7900HT real-time PCR system (from Applied Biosystems) in accordance with a protocol provided by Applied Biosystems.
  • RT Master Mix Perfect Real Time
  • the C5 mRNA expression level in the case that Hep3B cells were treated only with the transfection reagent without addition of siRNA was defined as 100%, and a C5 mRNA residual rate (relative value) was calculated for each introduction of siRNA by using a calibration curve method. As a negative control, Mock which does not cross over with any human gene was used.
  • Sense strands and antisense strands listed in Table 4 were synthesized by using the phosphoramidite method, and then annealed to synthesize double-stranded nucleic acids (GeneDesign, Inc.).
  • Double strand ID Numbers of nucleotides in sense strand/antisense strand Sense strand Antisense strand Sequence (5' ⁇ 3) SEQ ID NO Sequence (5' ⁇ 3') SEQ ID NO siRNA-001 21/21 AGGcAAAGGuGuucAAAGAdT ⁇ dT 89 UCUUUGAAcACCUUUGCCUdT ⁇ dT 90 siRNA-001-02 19/19 AGGcAAAGGuGuucAAAGA 91 UCUUUGAAcACCUUUGCCU 92 siRNA-001-08 21/21 A ⁇ GGcAAAGGuGuucAAAGAuu 93 U ⁇ CUUUGAAcACCUUUGCCUuu 94 siRNA-001-09 21/21 A ⁇ G ⁇ GcAAAGGu
  • Example 1 A test was performed to measure Ct values for the target gene human C5 and the endogenous control gene human GAPDH in cultured Hep3B cells in the same manner as in Example 1, except that siRNA/RNAiMax mixed solution was prepared with a final concentration of 1 nM double-stranded nucleic acid and 0.3% RNAiMax.
  • the C5 mRNA expression level in the case of Lipofection only was defined as 100%, and a C5 mRNA residual rate (relative value) was calculated for each introduction of siRNA.
  • siRNA listed in Table 6 was dissolved in 10 mM sodium citrate (pH 4.0) to prepare diluted siRNA solution.
  • Lipid solution was prepared by dissolving 2- ⁇ 9-oxo-9-[(3-pentyloctyl)oxy]nonyl ⁇ dodecyl 1-methylpiperidine-4-carboxylate, DSPC (NIPPON FINE CHEMICAL CO., LTD.), Cholesterol (NIPPON FINE CHEMICAL CO., LTD.), and MPEG2000-DMG (NOF CORPORATION) at a mole ratio of 60/10.5/28/1.5 in ethanol.
  • Lipid Nanoparticles were obtained by mixing the diluted siRNA solution and the lipid solution at flow rates of 3 mL/min and 1 mL/min, respectively, with an siRNA/lipid weight ratio of 0.1.
  • the external solution of the resulting LNP aqueous solution was substituted with PBS (pH 7.4) through dialysis by using a Float-A-Lyzer G2 (SPECTRUM, 100K MWCO). After the dialysis, the resultant was subjected to concentration and filtration sterilization for use in experiments.
  • the siRNA concentration and encapsulation efficiency were measured by using a Quant-iT RiboGreen RNA Reagent (Invitrogen, Cat#R11491).
  • the siRNA concentration measured after dilution with RNase Free Water was assumed as the concentration of siRNA present in the LNP external solution
  • the siRNA concentration measured after dilution with 1% Triton X-100 was assumed as the total siRNA concentration of the formulation.
  • the average particle size (Z-average) was measured by using a particle size analyzer (Malven Panalytical Ltd., Zetasizer Nano ZS). Results of evaluation of product quality for the prepared LNPs are shown in Table 7.
  • Ct values were measured for the target gene mouse C5 and the endogenous control gene mouse GAPDH by using a TaqMan (registered trademark) Gene Expression Master Mix (Applied Biosystem, Cat#4369510) and a TaqMan probe (Applied Biosystems, C5: Mm01336776_g1; GAPDH: Mm99999915_g1) with an ABI7500 Fast (Applied Biosystems) in accordance with a protocol provided by the manufacturer.
  • the liver C5 mRNA residual rate 5 days after the administration for the PBS administration group was defined as 100%, and a liver C5 mRNA residual rate (relative value) was calculated for each siRNA administration group by using the comparative Ct method. The results are shown in Table 8.
  • mice anti-C5 antibody BB5.1 Hycult Biotech, Cat#HM1073-FS
  • PBS(-) Wi-Fi Protected S-100
  • blocking solution PBS(-) (Wako Pure Chemical Industries, Ltd.) containing 1% BSA (R&D systems, Inc., Cat#DY995)
  • PBS(-) Wired Chemical Industries, Ltd.
  • BSA R&D systems, Inc., Cat#DY995
  • the washing solution was discarded, and the heparin plasma sample diluted with blocking solution was then added, and the resultant was incubated at room temperature for 5 hours.
  • the plasma of the PBS administration group was used as a standard sample.
  • the sample was discarded, and washing was then performed five times with washing solution, and a goat anti-human C5 antibody (Quidel Corporation, Cat#A306) diluted 4000-fold with blocking solution was added, and the resultant was incubated at room temperature for 1 hour.
  • the antibody was discarded, and washing was then performed five times with washing solution, and an HRP-labeled donkey anti-goat IgG (H+L) (Jackson ImmunoResearch Inc., Cat#805-035-180) diluted 40000-fold with blocking solution was added, and the resultant was incubated at room temperature for 1 hour.
  • the antibody was discarded, and washing was then performed five times with washing solution.
  • Sense strands and antisense strands listed in Table 10 were synthesized by using the phosphoramidite method, and then annealed to synthesize double-stranded nucleic acids (GeneDesign, Inc.). A test was performed to measure Ct values for the target gene human C5 and the endogenous control gene human GAPDH in cultured Hep3B cells in the same manner as in Example 1, except that siRNA/RNAiMax mixed solution was prepared with a final concentration of 0.003 to 10 nM double-stranded nucleic acid and 0.3% RNAiMax.
  • Lipid nanoparticles (LNPs) encapsulating siRNA therein were prepared in the same manner as in Example 3, except that siRNAs listed in Table 12 were used. Results of evaluation of product quality for the prepared LNPs are shown in Table 13.
  • Table 12 Double strand ID Numbers of nucleotides in sense strand/antisense strand Sense strand Antisense strand Sequence (5' ⁇ 3) SEQ ID NO Sequence (5' ⁇ 3) SEQ ID NO Mock 21/21 cuuAcGcuGAGuAcuucGAdT ⁇ dT 87 UCGAAGuACUcAGCGuAAGdT ⁇ dT 88 siRNA-008 21/21 uGGuAuAuGuGuuGcuGAudT ⁇ dT 13 AUcAGcAAcAcAuAuACcAdT ⁇ dT 14 siRNA-008-32 19/23 uGGuAuAuGuGuuGCuGAu 141 AUcAGcAAcAcAuAuA
  • Total RNA was purified by using an RNeasy Plus Mini Kit (Qiagen, Cat#74106) in accordance with a protocol provided by the manufacturer.
  • cDNA was prepared by using a PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc., Cat#RR036A) in accordance with a protocol provided by the manufacturer.
  • Ct values were measured for the target gene mouse C5 and the endogenous control gene mouse GAPDH by using a TaqMan (registered trademark) Gene Expression Master Mix (Applied Biosystem, Cat#4369510) and a TaqMan probe (Applied Biosystems, C5: Mm01336776_g1; GAPDH: Mm99999915_g1) with an ABI7500 Fast (Applied Biosystems) in accordance with a protocol provided by the manufacturer.
  • the liver C5 mRNA residual rate on each day of measurement for the PBS administration group was defined as 100%, and liver C5 mRNA residual rates (relative values) were calculated for each siRNA administration group by using the comparative Ct method. The results are shown in Table 14.
  • mice anti-C5 antibody BB5.1 Hycult Biotech, Cat#HM1073-FS
  • PBS(-) Wi-Fi Protected S-100
  • blocking solution PBS(-) (Wako Pure Chemical Industries, Ltd.) containing 1% BSA (R&D systems, Inc., Cat#DY995)
  • PBS(-) Wired Chemical Industries, Ltd.
  • BSA R&D systems, Inc., Cat#DY995
  • the washing solution was discarded, and the heparin plasma sample diluted with blocking solution was then added, and the resultant was incubated at room temperature for 5 hours.
  • the plasma of the PBS administration group was used as a standard sample.
  • the sample was discarded, and washing was then performed five times with washing solution, and a goat anti-human C5 antibody (Quidel Corporation, Cat#A306) diluted 4000-fold with blocking solution was added, and the resultant was incubated at room temperature for 1 hour.
  • the antibody was discarded, and washing was then performed five times with washing solution, and an HRP-labeled donkey anti-goat IgG (H+L) (Jackson ImmunoResearch Inc., Cat#805-035-180) diluted 40000-fold with blocking solution was added, and the resultant was incubated at room temperature for 1 hour.
  • the antibody was discarded, and washing was then performed five times with washing solution.
  • Liver C5 mRNA residual rates and plasma C5 concentrations 5 days, 14 days, and 21 days after the administration were quantified, and subjected to statistical analysis (unpaired T-test) for the siRNA-008-34 administration group to the siRNA-008 administration group.
  • the results are shown in FIG. 1 and FIG. 2 .
  • Groups with a P value of 0.05 or lower were provided with * (asterisk), and groups with a P value of 0.01 or lower were provided with **.
  • Sense strands and antisense strands listed in Table 16 were synthesized by using the phosphoramidite method, and then annealed to synthesize double-stranded nucleic acids (GeneDesign, Inc.). As in Example 1, the C5 mRNA expression level in the case of Lipofection only was defined as 100%, and a C5 mRNA residual rate (relative value) was calculated for each introduction of siRNA. The results are shown in Table 17.
  • LNPs Lipid nanoparticles encapsulating siRNA therein were prepared in the same manner as in Example 3, except that siRNAs listed in Table 18 were used. Results of evaluation of product quality for the prepared LNPs are shown in Table 19.
  • Table 18 Double strand ID Numbers of nucleotides in sense strand/antisense strand Sense strand Antisense strand Sequence (5' ⁇ 3') SEQ ID NO Sequence (5' ⁇ 3') SEQ ID NO Mock 21/21 cuuAcGcuGAGuAcuucGAdT ⁇ dT 89 UCGAAGuACUcAGCGuAAGdT ⁇ dT 90 siRNA-008-34 19/23 uGGuAuAuGuGuuGCuGAu 145 AUcAGcAAcAcAuAuACcAuu ⁇ a ⁇ a 146 [Table 19] Double strand ID Encapsulation efficiency Average particle size (nm) Polydispersity index Mock >90% 92 0.06 siRNA-008-34 >
  • the blood sampled on each sampling day was placed in a blood separator tube containing clot activator (Immuno-Biological Laboratories Co, Ltd., Cat#31203) and centrifuged at 3000 rpm for 15 minutes, and then the serum as the supernatant was collected and stored at -80°C. Thereafter, the complement activity in the serum was quantified in the following manner.
  • sheep erythrocytes with a concentration of 1.5 ⁇ 10 8 cells/mL were prepared by using a serum complement titer CH50 kit (DENKA SEIKEN Co., Ltd., Cat#400017) in accordance with a protocol provided by the manufacturer.
  • zymosan (Wako Pure Chemical Industries, Ltd., Cat#263-01491) was prepared so as at a dose of 20 ⁇ g/mL with a diluting medium attached to the serum complement titer CH50 kit.
  • the sample serum was diluted 40-fold with the same diluting medium.
  • the sheep erythrocytes, the zymosan, and the diluted serum sample each in a volume of 50 ⁇ L were mixed together, and the mixture was incubated at 37°C overnight. On the next day, the assay plate was centrifuged at 2000 rpm at room temperature for 10 minutes, and the absorbance of the supernatant was then measured at 405 nm. Values for the samples as the complement activity in the serum on the day before the administration to each individual was defined as 100% are shown in Table 20.
  • Lipid nanoparticles (LNPs) encapsulating siRNA therein were prepared in the same manner as in Example 3, except that siRNAs listed in Table 21 were used. Results of evaluation of product quality for the prepared LNPs are shown in Table 22.
  • Table 21 Double strand ID Numbers of nucleotides in sense strand/antisense strand Sense strand Antisense strand Sequence (5' ⁇ 3) SEQ ID NO Sequence (5' ⁇ 3') SEQ ID NO siRNA-008-34 19/23 uGGuAuAuGuGuuGCuGAu 145 AUcAGcAAcAcAuuAuACcAuu ⁇ a ⁇ a 146
  • the blood sampled on each sampling day was placed in a blood separator tube containing clot activator (Immuno-Biological Laboratories Co, Ltd., Cat#31203) and centrifuged at 3000 rpm for 15 minutes, and then the serum as the supernatant was collected and stored at -80°C. Thereafter, the complement activity in the serum was quantified in the following manner. Specifically, sheep erythrocytes with a concentration of 1.5 ⁇ 10 8 cells/mL were prepared by using a serum complement titer CH50 kit (DENKA SEIKEN Co., Ltd., Cat#400017) in accordance with a protocol provided by the manufacturer.
  • zymosan (Wako Pure Chemical Industries, Ltd., Cat#263-01491) was prepared so as at a dose of 20 ⁇ g/mL with a diluting medium attached to the serum complement titer CH50 kit.
  • the sample serum was diluted 40-fold with the same diluting medium.
  • the sheep erythrocytes, the zymosan, and the diluted serum sample each in a volume of 50 ⁇ L were mixed together, and the mixture was incubated at 37°C overnight.
  • the assay plate was centrifuged at 2000 rpm at room temperature for 10 minutes, and the absorbance of the supernatant was then measured at 405 nm.
  • Lipid nanoparticles (LNPs) encapsulating siRNA therein were prepared in the same manner as in Example 8.
  • the blood was sampled under anesthesia on the day before the administration (-1 Day in Table 24), and 7 days, 13 days, 20 days and 27 days after the administration (7 Day, 13 Day, 20 Day and 27 Day in Table 24).
  • the blood sampled on each sampling day was placed in a blood separator tube containing clot activator (Immuno-Biological Laboratories Co, Ltd., Cat#31203) and centrifuged at 3000 rpm for 15 minutes, and then the serum as the supernatant was collected and stored at -80°C. Thereafter, the complement activity in the serum was quantified in the following manner. Specifically, sheep erythrocytes with a concentration of 1.5 ⁇ 10 8 cells/mL were prepared by using a serum complement titer CH50 kit (DENKA SEIKEN Co., Ltd., Cat#400017) in accordance with a protocol provided by the manufacturer.
  • zymosan (Wako Pure Chemical Industries, Ltd., Cat#263-01491) was prepared so as at a dose of 20 ⁇ g/mL with a diluting medium attached to the serum complement titer CH50 kit.
  • the sample serum was diluted 40-fold with the same diluting medium.
  • the sheep erythrocytes, the zymosan, and the diluted serum sample each in a volume of 50 ⁇ L were mixed together, and the mixture was incubated at 37°C overnight.
  • the assay plate was centrifuged at 2000 rpm at room temperature for 10 minutes, and the absorbance of the supernatant was then measured at 405 nm.
  • Lipid nanoparticles (LNPs) encapsulating siRNA therein were prepared in the same manner as in Example 8.
  • the blood was sampled under anesthesia on the day before the administration (-1 Day in Table 25), and 7 days, 13 days, 20 days, 27 days, 34 days, 41 days, 48 days and 55 days after the administration (7 Day, 13 Day, 20 Day, 27 Day, 34 Day, 41 Day, 48 Day and 55 Day in Table 25).
  • the blood sampled on each sampling day was placed in a blood separator tube containing clot activator (Immuno-Biological Laboratories Co, Ltd., Cat#31203) and centrifuged at 3000 rpm for 15 minutes, and then the serum as the supernatant was collected and stored at -80°C. Thereafter, the complement activity in the serum was quantified in the following manner. Specifically, sheep erythrocytes with a concentration of 1.5 ⁇ 10 8 cells/mL were prepared by using a serum complement titer CH50 kit (DENKA SEIKEN Co., Ltd., Cat#400017) in accordance with a protocol provided by the manufacturer.
  • zymosan (Wako Pure Chemical Industries, Ltd., Cat#263-01491) was prepared so as at a dose of 20 ⁇ g/mL with a diluting medium attached to the serum complement titer CH50 kit.
  • the sample serum was diluted 40-fold with the same diluting medium.
  • the sheep erythrocytes, the zymosan, and the diluted serum sample each in a volume of 50 ⁇ L were mixed together, and the mixture was incubated at 37°C overnight.
  • the assay plate was centrifuged at 2000 rpm at room temperature for 10 minutes, and the absorbance of the supernatant was then measured at 405 nm.
  • Lipid nanoparticles (LNPs) encapsulating siRNA therein were prepared in the same manner as in Example 8.
  • the blood was sampled under anesthesia on the day before the administration (-1 Day in Table 26), and 7 days, 13 days, 20 days, 27 days, 34 days, 41 days, 48 days and 55 days after the administration (7 Day, 13 Day, 20 Day, 27 Day, 34 Day, 41 Day, 48 Day and 55 Day in Table 26).
  • the blood sampled on each sampling day was placed in a blood separator tube containing clot activator (Immuno-Biological Laboratories Co, Ltd., Cat#31203) and centrifuged at 3000 rpm for 15 minutes, and then the serum as the supernatant was collected and stored at -80°C. Thereafter, the complement activity in the serum was quantified in the following manner. Specifically, sheep erythrocytes with a concentration of 1.5 ⁇ 10 8 cells/mL were prepared by using a serum complement titer CH50 kit (DENKA SEIKEN Co., Ltd., Cat#400017) in accordance with a protocol provided by the manufacturer.
  • zymosan (Wako Pure Chemical Industries, Ltd., Cat#263-01491) was prepared so as at a dose of 20 ⁇ g/mL with a diluting medium attached to the serum complement titer CH50 kit.
  • the sample serum was diluted 40-fold with the same diluting medium.
  • the sheep erythrocytes, the zymosan, and the diluted serum sample each in a volume of 50 ⁇ L were mixed together, and the mixture was incubated at 37°C overnight.
  • the assay plate was centrifuged at 2000 rpm at room temperature for 10 minutes, and the absorbance of the supernatant was then measured at 405 nm.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Diabetes (AREA)
  • Urology & Nephrology (AREA)
  • Dispersion Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Anti-Oxidant Or Stabilizer Compositions (AREA)
  • Cosmetics (AREA)
  • Dc Digital Transmission (AREA)
  • Medicinal Preparation (AREA)
  • Saccharide Compounds (AREA)
EP19876529.9A 2018-10-26 2019-10-24 Double-stranded ribonucleic acid inhibiting expression of complement c5 Active EP3871680B1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2018201777 2018-10-26
US16/354,916 US10526603B1 (en) 2018-10-26 2019-03-15 Double-stranded ribonucleic acid capable of suppressing expression of complement C5
PCT/JP2019/041783 WO2020085456A1 (ja) 2018-10-26 2019-10-24 補体ｃ５の発現を抑制する二本鎖リボ核酸

Publications (3)

Publication Number Publication Date
EP3871680A1 EP3871680A1 (en) 2021-09-01
EP3871680A4 EP3871680A4 (en) 2022-09-21
EP3871680B1 true EP3871680B1 (en) 2024-03-20

Family

ID=69058781

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19876529.9A Active EP3871680B1 (en) 2018-10-26 2019-10-24 Double-stranded ribonucleic acid inhibiting expression of complement c5

Country Status (20)

Country Link
US (1) US10526603B1 (zh)
EP (1) EP3871680B1 (zh)
JP (1) JP6725776B1 (zh)
KR (1) KR20210086608A (zh)
CN (1) CN112771163B (zh)
AR (1) AR116832A1 (zh)
AU (1) AU2019367536A1 (zh)
BR (1) BR112021006520A2 (zh)
CA (1) CA3115249A1 (zh)
CL (2) CL2021000826A1 (zh)
CO (1) CO2021004029A2 (zh)
IL (1) IL281902B (zh)
JO (1) JOP20210066A1 (zh)
MX (1) MX2021003910A (zh)
PE (1) PE20210811A1 (zh)
PH (1) PH12021550736A1 (zh)
SG (1) SG11202102881PA (zh)
TW (2) TWI718717B (zh)
WO (1) WO2020085456A1 (zh)
ZA (1) ZA202102104B (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220122608A (ko) * 2019-12-26 2022-09-02 에자이 알앤드디 매니지먼트 가부시키가이샤 보체 c5의 발현을 저해하는 이중 가닥 리보핵산을 함유하는 약제학적 조성물
JP6918197B2 (ja) * 2019-12-26 2021-08-11 エーザイ・アール・アンド・ディー・マネジメント株式会社 脂質複合体を含む医薬組成物及び脂質ナノ粒子を含む医薬組成物
WO2021154941A1 (en) * 2020-01-31 2021-08-05 Alnylam Pharmaceuticals, Inc. Complement component c5 irna compositions for use in the treatment of amyotrophic lateral sclerosis (als)
WO2023245126A2 (en) * 2022-06-15 2023-12-21 Sirnaomics, Inc. Products and compositions
WO2024169908A1 (zh) * 2023-02-17 2024-08-22 苏州时安生物技术有限公司 一种调节补体C5表达的siRNA、其缀合物和药物组合物及用途

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2904654C (en) 2013-03-14 2023-12-05 Alnylam Pharmaceuticals, Inc. Complement component c5 irna compositions and methods of use thereof
US9873669B2 (en) 2014-01-09 2018-01-23 Eisai R&D Management Co., Ltd. Cationic lipid
EP3194596A1 (en) * 2014-09-16 2017-07-26 Alnylam Pharmaceuticals, Inc. Complement component c5 irna compositions and methods of use thereof
JP6592458B2 (ja) 2014-12-26 2019-10-16 エーザイ・アール・アンド・ディー・マネジメント株式会社 カチオン性脂質
US10036017B2 (en) 2015-02-17 2018-07-31 Dicerna Pharmaceuticals, Inc. Methods and compositions for the specific inhibition of complement component 5(C5) by double-stranded RNA
EP3307316A1 (en) * 2015-06-12 2018-04-18 Alnylam Pharmaceuticals, Inc. Complement component c5 irna compositions and methods of use thereof
JP2019518028A (ja) * 2016-06-10 2019-06-27 アルナイラム ファーマシューティカルズ， インコーポレイテッドＡｌｎｙｌａｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 補体成分Ｃ５ｉＲＮＡ組成物及び発作性夜間血色素尿症（ＰＮＨ）を処置するためのその使用方法
SG11201808928YA (en) 2016-06-24 2018-11-29 Eisai R&D Man Co Ltd Cationic lipid

Also Published As

Publication number Publication date
WO2020085456A1 (ja) 2020-04-30
JOP20210066A1 (ar) 2023-01-30
JP6725776B1 (ja) 2020-07-22
CL2023001965A1 (es) 2023-12-15
IL281902B (en) 2022-09-01
IL281902A (en) 2021-05-31
CN112771163A (zh) 2021-05-07
KR20210086608A (ko) 2021-07-08
PE20210811A1 (es) 2021-04-28
JPWO2020085456A1 (ja) 2021-02-15
BR112021006520A2 (pt) 2021-07-06
SG11202102881PA (en) 2021-04-29
EP3871680A1 (en) 2021-09-01
AR116832A1 (es) 2021-06-16
CN112771163B (zh) 2024-07-12
TW202031269A (zh) 2020-09-01
PH12021550736A1 (en) 2021-10-25
MX2021003910A (es) 2021-06-04
AU2019367536A1 (en) 2021-04-29
TW202135833A (zh) 2021-10-01
ZA202102104B (en) 2022-08-31
TWI818225B (zh) 2023-10-11
EP3871680A4 (en) 2022-09-21
TWI718717B (zh) 2021-02-11
US10526603B1 (en) 2020-01-07
CO2021004029A2 (es) 2021-04-30
CL2021000826A1 (es) 2021-09-10
CA3115249A1 (en) 2020-04-30

Similar Documents

Publication Publication Date Title
EP3871680B1 (en) Double-stranded ribonucleic acid inhibiting expression of complement c5
EP1842558B1 (en) Composition for inhibiting expression of target gene
DE112020003843T5 (de) Verbesserte Lipid-Nanopartikel zur Zuführung von Nukleinsäuren
US20220136011A1 (en) Telomerase-containing exosomes for treatment of diseases associated with aging and age-related organ dysfunction
TW200911989A (en) RNAi inhibition of alpha-ENaC expression
WO2017135397A1 (ja) 補体ｂ因子の発現を抑制するアンチセンスオリゴヌクレオチド
WO2024002320A1 (zh) 一种抑制b7-h3基因表达的干扰rna及其应用
WO2018117253A1 (ja) 補体ｂ因子の発現を抑制する核酸
EP3594345A1 (en) Nucleic acid capable of inhibiting expression of masp2
EP4052710A1 (en) Pharmaceutical composition containing double-stranded ribonucleic acid inhibiting expression of complement c5
RU2781954C1 (ru) Двухцепочечная рибонуклеиновая кислота, ингибирующая экспрессию компонента комплемента c5
US20230092306A1 (en) Substance delivery carrier and composition
JP6918197B2 (ja) 脂質複合体を含む医薬組成物及び脂質ナノ粒子を含む医薬組成物
EP3095867A1 (en) Nucleic acid capable of inhibiting expression of beta2gpi
TW201903149A (zh) 抑制apcs之表現的核酸
WO2024129826A2 (en) Compositions and methods for modulating hsp70 activity
WO2024026565A1 (en) Compositions and methods for inhibiting adenylate cyclase 9 (ac9)

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20210401

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Ref document number: 602019048790

Country of ref document: DE

Free format text: PREVIOUS MAIN CLASS: A61K0031713000

Ipc: C12N0015113000

Ref country code: DE

Ref legal event code: R079

Free format text: PREVIOUS MAIN CLASS: A61K0031713000

Ipc: C12N0015113000

A4 Supplementary search report drawn up and despatched

Effective date: 20220819

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 7/00 20060101ALN20220815BHEP

Ipc: A61K 31/7125 20060101ALI20220815BHEP

Ipc: A61K 31/712 20060101ALI20220815BHEP

Ipc: A61K 47/56 20170101ALI20220815BHEP

Ipc: A61K 47/54 20170101ALI20220815BHEP

Ipc: A61K 47/34 20170101ALI20220815BHEP

Ipc: A61K 47/28 20060101ALI20220815BHEP

Ipc: A61K 47/24 20060101ALI20220815BHEP

Ipc: A61K 47/22 20060101ALI20220815BHEP

Ipc: A61K 9/127 20060101ALI20220815BHEP

Ipc: A61P 43/00 20060101ALI20220815BHEP

Ipc: A61P 13/00 20060101ALI20220815BHEP

Ipc: A61K 31/713 20060101ALI20220815BHEP

Ipc: C12N 15/113 20100101AFI20220815BHEP

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230511

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 7/00 20060101ALN20230810BHEP

Ipc: A61K 31/7125 20060101ALI20230810BHEP

Ipc: A61K 31/712 20060101ALI20230810BHEP

Ipc: A61K 47/56 20170101ALI20230810BHEP

Ipc: A61K 47/54 20170101ALI20230810BHEP

Ipc: A61K 47/34 20170101ALI20230810BHEP

Ipc: A61K 47/28 20060101ALI20230810BHEP

Ipc: A61K 47/24 20060101ALI20230810BHEP

Ipc: A61K 47/22 20060101ALI20230810BHEP

Ipc: A61K 9/127 20060101ALI20230810BHEP

Ipc: A61P 43/00 20060101ALI20230810BHEP

Ipc: A61P 13/00 20060101ALI20230810BHEP

Ipc: A61K 31/713 20060101ALI20230810BHEP

Ipc: C12N 15/113 20100101AFI20230810BHEP

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 7/00 20060101ALN20230811BHEP

Ipc: A61K 31/7125 20060101ALI20230811BHEP

Ipc: A61K 31/712 20060101ALI20230811BHEP

Ipc: A61K 47/56 20170101ALI20230811BHEP

Ipc: A61K 47/54 20170101ALI20230811BHEP

Ipc: A61K 47/34 20170101ALI20230811BHEP

Ipc: A61K 47/28 20060101ALI20230811BHEP

Ipc: A61K 47/24 20060101ALI20230811BHEP

Ipc: A61K 47/22 20060101ALI20230811BHEP

Ipc: A61K 9/127 20060101ALI20230811BHEP

Ipc: A61P 43/00 20060101ALI20230811BHEP

Ipc: A61P 13/00 20060101ALI20230811BHEP

Ipc: A61K 31/713 20060101ALI20230811BHEP

Ipc: C12N 15/113 20100101AFI20230811BHEP

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 7/00 20060101ALN20230830BHEP

Ipc: A61K 31/7125 20060101ALI20230830BHEP

Ipc: A61K 31/712 20060101ALI20230830BHEP

Ipc: A61K 47/56 20170101ALI20230830BHEP

Ipc: A61K 47/54 20170101ALI20230830BHEP

Ipc: A61K 47/34 20170101ALI20230830BHEP

Ipc: A61K 47/28 20060101ALI20230830BHEP

Ipc: A61K 47/24 20060101ALI20230830BHEP

Ipc: A61K 47/22 20060101ALI20230830BHEP

Ipc: A61K 9/127 20060101ALI20230830BHEP

Ipc: A61P 43/00 20060101ALI20230830BHEP

Ipc: A61P 13/00 20060101ALI20230830BHEP

Ipc: A61K 31/713 20060101ALI20230830BHEP

Ipc: C12N 15/113 20100101AFI20230830BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 7/00 20060101ALN20230907BHEP

Ipc: A61K 31/7125 20060101ALI20230907BHEP

Ipc: A61K 31/712 20060101ALI20230907BHEP

Ipc: A61K 47/56 20170101ALI20230907BHEP

Ipc: A61K 47/54 20170101ALI20230907BHEP

Ipc: A61K 47/34 20170101ALI20230907BHEP

Ipc: A61K 47/28 20060101ALI20230907BHEP

Ipc: A61K 47/24 20060101ALI20230907BHEP

Ipc: A61K 47/22 20060101ALI20230907BHEP

Ipc: A61K 9/127 20060101ALI20230907BHEP

Ipc: A61P 43/00 20060101ALI20230907BHEP

Ipc: A61P 13/00 20060101ALI20230907BHEP

Ipc: A61K 31/713 20060101ALI20230907BHEP

Ipc: C12N 15/113 20100101AFI20230907BHEP

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 7/00 20060101ALN20230914BHEP

Ipc: A61K 31/7125 20060101ALI20230914BHEP

Ipc: A61K 31/712 20060101ALI20230914BHEP

Ipc: A61K 47/56 20170101ALI20230914BHEP

Ipc: A61K 47/54 20170101ALI20230914BHEP

Ipc: A61K 47/34 20170101ALI20230914BHEP

Ipc: A61K 47/28 20060101ALI20230914BHEP

Ipc: A61K 47/24 20060101ALI20230914BHEP

Ipc: A61K 47/22 20060101ALI20230914BHEP

Ipc: A61K 9/127 20060101ALI20230914BHEP

Ipc: A61P 43/00 20060101ALI20230914BHEP

Ipc: A61P 13/00 20060101ALI20230914BHEP

Ipc: A61K 31/713 20060101ALI20230914BHEP

Ipc: C12N 15/113 20100101AFI20230914BHEP

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 7/00 20060101ALN20230921BHEP

Ipc: A61K 31/7125 20060101ALI20230921BHEP

Ipc: A61K 31/712 20060101ALI20230921BHEP

Ipc: A61K 47/56 20170101ALI20230921BHEP

Ipc: A61K 47/54 20170101ALI20230921BHEP

Ipc: A61K 47/34 20170101ALI20230921BHEP

Ipc: A61K 47/28 20060101ALI20230921BHEP

Ipc: A61K 47/24 20060101ALI20230921BHEP

Ipc: A61K 47/22 20060101ALI20230921BHEP

Ipc: A61K 9/127 20060101ALI20230921BHEP

Ipc: A61P 43/00 20060101ALI20230921BHEP

Ipc: A61P 13/00 20060101ALI20230921BHEP

Ipc: A61K 31/713 20060101ALI20230921BHEP

Ipc: C12N 15/113 20100101AFI20230921BHEP

INTG Intention to grant announced

Effective date: 20231005

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602019048790

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG9D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240621

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240620

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20240320

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240620

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240620

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240621

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1667846

Country of ref document: AT

Kind code of ref document: T

Effective date: 20240320

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240720

VS25 Lapsed in a validation state [announced via postgrant information from nat. office to epo]

Ref country code: MD

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240320

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240722